Receptor Subunit Research Articles

Cheonwangbosimdan mitigates post-traumatic stress disorder-like behaviors through GluN2B-containing NMDA receptor antagonism in mice

Ethnopharmacological relevanceCheonwangbosimdan (CWBSD), a herbal medicine traditionally used for anxiety, insomnia, depression, and heart palpitations, has been reported to have anti-anxiety, antidepressant, cognitive improvement, and neuroprotective effects. Aim of the studyThe purpose of this study was to determine if CWBSD could affect post-traumatic stress disorder (PTSD)-like behaviors because it has prioritized clinical use over mechanism study. Materials and methodsA single prolonged stress (SPS) mouse model, a well-established animal model of PTSD, was used to investigate whether standardized CWBSD could mitigate PTSD-like behaviors through robust behavioral tests, including the elevated plus-maze test and marble burying test for measuring anxiety-like behaviors, the splash test, forced swimming test, and tail suspension test for evaluating depression-like behaviors, and the Y-maze test and novel object recognition test for assessing cognitive function. Additionally, a fear extinction test was employed to determine whether CWBSD might reverse fear memory extinction deficits. Amygdala tissue was isolated from SPS-treated mouse brain and subjected to Western blotting or quantitative PCR to explore mechanisms by which CWBSD could mitigate PTSD-like behaviors. ResultsCWBSD ameliorated emotional impairments and cognitive dysfunction in an SPS-induced PTSD-like mouse model. It also mitigated deficits in abnormal fear memory extinction. Protein expression levels of N-methyl-D-aspartate (NMDA) receptor subunit 2B (GluN2B) and phosphorylation levels of Ca2+/calmodulin-dependent protein kinase II in the amygdala were increased in SPS model mice and normalized by CWBSD. Additionally, co-administration of CWBSD and GluN2B-containing NMDA receptor antagonist, ifenprodil, at each sub-effective dose promoted fear memory extinction. ConclusionsCWBSD can alleviate SPS-induced PTSD-like behaviors by normalizing GluN2B-containing NMDA receptor activity in the amygdala. Therefore, CWBSD could be a promising candidate for PTSD treatment with fewer adverse effects and better efficacy than existing therapies.

Antinociceptive Effects of α2/α3-Subtype-Selective GABAA Receptor Positive Allosteric Modulators KRM-II-81 and NS16085 in Male Rats: Behavioral Specificity.

Recent studies suggest that among the gamma-aminobutyric acid type A (GABAA)receptor subtype heterogeneity, α2/α3 subunits of GABAA receptors mediate pain processing. Therefore, α2/α3 subtype-selective GABAA receptor-positive allosteric modulators (PAMs) may be candidate analgesics. Antinociceptive effects of α2/α3 subtype-selective GABAA receptor PAMs have been reported, but the behavioral effects of these compounds have not been systematically evaluated. This study examined the behavioral effects of two α2/α3 subtype-selective GABAA receptor PAMs, KRM-II-81 and NS16085, in male rats. The antinociceptive effects of KRM-II-81 and NS16085 were examined using rat models of inflammatory (complete Freund's adjuvant) and neuropathic pain (chronic constriction injury). The effect of KRM-II-81 on affective pain was measured using the place escape/avoidance paradigm (PEAP). Rate-response of food-maintained operant responding, horizontal wire test, and the spontaneous alternation T-maze were assessed to study the side-effect profiles of KRM-II-81 and NS16085. The benzodiazepine midazolam was used as a comparator in these studies. KRM-II-81 and NS16085 attenuated mechanical allodynia but not thermal hyperalgesia in both pain states, and their effects were attenuated by the benzodiazepine receptor antagonist flumazenil. KRM-II-81 attenuated affective pain-related behavior in the PEAP test. In the operant responding procedure and horizontal wire test, only midazolam produced significant effects at the dose that produced maximal antinociception. In the T-maze assay, only midazolam significantly decreased the percentage of alternation at an antinociceptive dose. Thus, KRM-II-81 and NS16085 but not midazolam selectively produced antinociceptive effects. Collectively, these data suggest that α2/α3 subtype-selective GABAA PAMs could be a novel class of analgesics and warrant further investigation. SIGNIFICANCE STATEMENT: This study demonstrates that α2/α3 subtype-selective GABAA PAMs KRM-II-81 and NS16085 produce selective antinociceptive effects devoid of sedation, myorelaxation, and cognitive impairment in two rat models of persistent pain. This study supports the development of α2/α3 subtype-selective GABAA PAMs, rather than classical benzodiazepines, as safe and novel analgesics for pain management.

Changes in ethanol effects in knock-in mice expressing ethanol insensitive alpha1 and alpha2 glycine receptor subunits

AimsGlycine receptors (GlyRs) are potentiated by physiologically relevant concentrations of ethanol, and mutations in the intracellular loop of α1 and α2 subunits reduced the effect of the drug. Knock-in (KI) mice having these individual mutations revealed that α1 and α2 subunits played a role in ethanol-induced sedation and ethanol intake. In this study, we wanted to examine if the effects of stacking both mutations in a 2xKI mouse model (α1/α2) generated by a selective breeding strategy further impacted cellular and behavioral responses to ethanol. Main methodsWe used electrophysiological recordings to examine ethanol's effect on GlyRs and evaluated ethanol-induced neuronal activation using c-Fos immunoreactivity and the genetically encoded calcium indicator GCaMP6s in the nucleus accumbens (nAc). We also examined ethanol-induced behavior using open field, loss of the righting response, and drinking in the dark (DID) paradigm. Key findingsEthanol did not potentiate GlyRs nor affect neuronal excitability in the nAc from 2xKI. Moreover, ethanol decreased the Ca2+ signal in WT mice, whereas there were no changes in the signal in 2xKI mice. Interestingly, there was an increase in c-Fos baseline in the 2xKI mice in the absence of ethanol. Behavioral assays showed that 2xKI mice recovered faster from a sedative dose of ethanol and had higher ethanol intake on the first test day of the DID test than WT mice. Interestingly, an open-field assay showed that 2xKI mice displayed less anxiety-like behavior than WT mice. SignificanceThe results indicate that α1 and α2 subunits are biologically relevant targets for regulating sedative effects and ethanol consumption.

Dipeptidyl-peptidase 4 (DPP4) mediates fatty acid uptake inhibition by glucose via TAS1R3 and GLUT-2 in Caco-2 enterocytes

Both high glucose intake with a high-fat meal and inhibition of dipeptidyl peptidase-4 (DPP4) have been associated with plasma lipid-lowering effects, but mechanistic understanding linking glucose and fat absorption is lacking. We here hypothesized that glucose ameliorates intestinal fatty acid uptake via a pathway involving DPP4. A concentration of 50 mM glucose reduced mean DPP4 activity in differentiated Caco-2 enterocytes by 42.5 % and fatty acid uptake by 66.0 % via nutrient sensing by the sweet taste receptor subunit TAS1R3 and glucose transporter GLUT-2. No effect of the DPP4 substrates GLP-1 and GIP or of the cellular energy status on the reduced uptake of fatty acids was seen, but a direct interaction between DPP4 and fatty acid transporters is suggested. Conclusively we identified DPP4 as a regulator of fatty acid absorption in Caco-2 enterocytes that mediates the inhibition of intestinal fatty acid uptake by glucose via an interplay of GLUT-2 and TAS1R3.

Seizures exacerbate excitatory: inhibitory imbalance in Alzheimer's disease and 5XFAD mice.

Approximately 22% of Alzheimer's disease (AD) patients suffer from seizures, and the co-occurrence of seizures and epileptiform activity exacerbates AD pathology and related cognitive deficits, suggesting that seizures may be a targetable component of AD progression. Given that alterations in neuronal excitatory:inhibitory (E:I) balance occur in epilepsy, we hypothesized that decreased markers of inhibition relative to those of excitation would be present in AD patients. We similarly hypothesized that in 5XFAD mice, the E:I imbalance would progress from an early stage (prodromal) to later symptomatic stages and be further exacerbated by pentylenetetrazol (PTZ) kindling. Post-mortem AD temporal cortical tissues from patients with or without seizure history were examined for changes in several markers of E:I balance, including levels of the inhibitory GABAA receptor, the sodium potassium chloride cotransporter 1 (NKCC1) and potassium chloride cotransporter 2 (KCC2) and the excitatory NMDA and AMPA type glutamate receptors. We performed patch-clamp electrophysiological recordings from CA1 neurons in hippocampal slices and examined the same markers of E:I balance in prodromal 5XFAD mice. We next examined 5XFAD mice at chronic stages, after PTZ or control protocols, and in response to chronic mTORC1 inhibitor rapamycin, administered following kindled seizures, for markers of E:I balance. We found that AD patients with comorbid seizures had worsened cognitive and functional scores and decreased GABAA receptor subunit expression, as well as increased NKCC1/KCC2 ratios, indicative of depolarizing GABA responses. Patch clamp recordings of prodromal 5XFAD CA1 neurons showed increased intrinsic excitability, along with decreased GABAergic inhibitory transmission and altered glutamatergic neurotransmission, indicating that E:I imbalance may occur in early disease stages. Furthermore, seizure induction in prodromal 5XFAD mice led to later dysregulation of NKCC1/KCC2 and a reduction in GluA2 AMPA glutamate receptor subunit expression, indicative of depolarizing GABA receptors and calcium permeable AMPA receptors. Finally, we found that chronic treatment with the mTORC1 inhibitor, rapamycin, at doses we have previously shown to attenuate seizure-induced amyloid-β pathology and cognitive deficits, could also reverse elevations of the NKCC1/KCC2 ratio in these mice. Our data demonstrate novel mechanisms of interaction between AD and epilepsy and indicate that targeting E:I balance, potentially with US Food and Drug Administration-approved mTOR inhibitors, hold therapeutic promise for AD patients with a seizure history.

Prophylactic treatment with the c-Abl inhibitor, neurotinib, diminishes neuronal damage and the convulsive state in pilocarpine-induced mice

The molecular mechanisms underlying seizure generation remain elusive, yet they are crucial for developing effective treatments for epilepsy. The current study shows that inhibiting c-Abl tyrosine kinase prevents apoptosis, reduces dendritic spine loss, and maintains N-methyl-d-aspartate (NMDA) receptor subunit 2B (NR2B) phosphorylated in invitro models of excitotoxicity. Pilocarpine-induced status epilepticus (SE) in mice promotes c-Abl phosphorylation, and disrupting c-Abl activity leads to fewer seizures, increases latency toward SE, and improved animal survival. Currently, clinically used c-Abl inhibitors are non-selective and have poor brain penetration. The allosteric c-Abl inhibitor, neurotinib, used here has favorable potency, selectivity, pharmacokinetics, and vastly improved brain penetration. Neurotinib-administered mice have fewer seizures and improved survival following pilocarpine-SE induction. Our findings reveal c-Abl kinase activation as a key factor in ictogenesis and highlight the impact of its inhibition in preventing the insurgence of epileptic-like seizures in rodents and humans.

Long-lasting increases in GABAB receptor subunit levels in hippocampal dentate gyrus of mice with a single systemic injection of trimethyltin

We have recently shown delayed increases in GABAB receptor (GABABR) subunit protein levels in the hippocampal dentate gyrus (DG), but not in the pyramidal CA1 and CA3 regions, at 15–30 days after the systemic single administration of trimethyltin (TMT) in mice. An attempt was thus made to determine whether the delayed increases return to the control levels found in naive mice afterward. In the DG on hippocampal slices obtained at 90 days after the administration, however, marked increases were still seen in protein levels of both GABABR1 and GABABR2 subunits without significant changes in calbindin and glial fibrillary acidic protein (GFAP) levels on immunoblotting analysis. Fluoro-Jade B staining clearly revealed the absence of degenerated neurons from the DG at 90 days after the administration. Although co-localization was invariably detected between GABABR2 subunit and GFAP in the DG at 30 days on immunohistochemical analysis, GABABR2-positive cells did not merge well with GFAP-positive cells in the DG at 90 days. These results suggest that both GABABR1 and GABABR2 subunits would be tardily and sustainably up-regulated by cells other than neurons and astrocytes in the murine DG at 90 days after a systemic single injection of TMT.

Blood eosinophilia in patients with severe bronchial asthma and chronic polypous rhinosinusitis treated with dupilumab: A review

Bronchial asthma and chronic polypous rhinosinusitis are diseases associated with a T2-inflammatory immune response. These nosologies can be combined, creating the preconditions for a more severe course of multimorbidity, requiring the use of genetic engineering biological therapy. Dupilumab is a monoclonal antibody that can specifically bind to the alpha subunit of the interleukin-4 receptor and block the action of interleukins 4 and 13, which play a key role in the development of T2 inflammation. Numerous studies have demonstrated the high effectiveness of this medicament. The use of dupilumab in some cases may be accompanied by an increase in eosinophils in the blood. This article presents scientific base and our own experience in treating patients with dupilumab-associated eosinophilia, in addition we describe an algorithm for examining this group of patients for the purpose of timely diagnosis of diseases such as eosinophilic granulomatosis with polyangiitis, eosinophilic pneumonia, etc. It should be noted that in the most cases eosinophilia during targeted therapy with dupilumab is temporary and does not cause clinical manifestations.

AAV-mediated upregulation of VDAC1 rescues the mitochondrial respiration and sirtuins expression in a SOD1 mouse model of inherited ALS.

Mitochondrial dysfunction represents one of the most common molecular hallmarks of both sporadic and familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder caused by the selective degeneration and death of motor neurons. The accumulation of misfolded proteins on and within mitochondria, as observed for SOD1 G93A mutant, correlates with a drastic reduction of mitochondrial respiration and the inhibition of metabolites exchanges, including ADP/ATP and NAD+/NADH, across the Voltage-Dependent Anion-selective Channel 1 (VDAC1), the most abundant channel protein of the outer mitochondrial membrane. Here, we show that the AAV-mediated upregulation of VDAC1 in the spinal cord of transgenic mice expressing SOD1 G93A completely rescues the mitochondrial respiratory profile. This correlates with the increased activity and levels of key regulators of mitochondrial functions and maintenance, namely the respiratory chain Complex I and the sirtuins (Sirt), especially Sirt3. Furthermore, the selective increase of these mitochondrial proteins is associated with an increase in Tom20 levels, the receptor subunit of the TOM complex. Overall, our results indicate that the overexpression of VDAC1 has beneficial effects on ALS-affected tissue by stabilizing the Complex I-Sirt3 axis.

Positive association between dehydroepiandrosterone (DHEA) and gene expression of the gamma-aminobutyric acid (GABA-A) receptor δ subunit

Gamma-aminobutyric acid A (GABA-A) receptors in the cells of the immune system enhance anti-inflammatory responses by regulating cytokine secretion, cytotoxic responses, and cell activation. In the CNS, the formation of GABA-A subunits into a pentameric structure has been extensively studied; however, no such study has been conducted in the immune system. The objective of the present study was to examine associations between the levels of steroid hormones and GABA-A receptor δ subunit expression in the immune system. We focused on this subunit because GABA-A receptors that contain it become significantly more sensitive to steroid hormones. We collected 80 blood samples from reproductive age women for the purpose of analyzing dehydroepiandrosterone (DHEA), 17β-estradiol, progesterone, and allopregnanolone using liquid chromatography-mass spectrometry (LC-MS). Furthermore, we extracted peripheral blood mononuclear cells (PBMCs) for determining mRNA expression levels of GABA-A receptor genes encoding the δ and ε subunits. We constructed linear mixed effect models for each GABA-A receptor subunit with all 4 steroid hormones, age, and age of menarche as predictors. Whereas DHEA was significantly associated with δ subunit expression (t-value = 2.981; p = 0.003), in line with our hypothesis, none of the steroid hormones were significantly associated with the expression of the ε subunit. Results of this study indicate that significant interactions between hormones from the steroid hormone biosynthesis pathway and GABAergic machinery from the immune cells may be utilized to expand models examining the molecular basis of inflammatory conditions.

Effects of Vaccination against Recombinant FSH or LH Receptor Subunits on Gonadal Development and Functioning Male Rats.

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) play key roles in regulating testosterone secretion and spermatogenesis in male mammals, respectively, and they maintain the fertility of male animals by binding to their corresponding receptors. We designed and prepared a recombinant LH receptor (LHR) subunit vaccine and a recombinant FSH receptor (FSHR) subunit vaccine and used male Sprague Dawley (SD) rats as a model to examine their effects on testicular development, spermatogenesis, and testosterone secretion in prepubertal and pubertal mammals. Both vaccines (LHR-DTT and FSHR-DTT) significantly decreased the serum testosterone level in prepubertal rats (p < 0.05) but had no effect on the testosterone secretion in pubertal rats; both vaccines decreased the number of cell layers in the seminiferous tubules and reduced spermatogenesis in prepubertal and pubertal rats. Subunit vaccine FSHR-DTT decreased the sperm density in the epididymis in both prepubertal and pubertal rats (p < 0.01) and lowered testicular index and sperm motility in pubertal rats (p < 0.05), whereas LHR-DTT only reduced the sperm density in the epididymis in pubertal rats (p < 0.05). These results indicate that the FSHR subunit vaccine may be a promising approach for immunocastration, but it still needs improvements in effectiveness.

Synaptic alterations associated with disrupted sensory encoding in a mouse model of tauopathy.

Synapse loss is currently the best biological correlate of cognitive decline in Alzheimer's disease and other tauopathies. Synapses seem to be highly vulnerable to tau-mediated disruption in neurodegenerative tauopathies. However, it is unclear how and when this leads to alterations in function related to the progression of tauopathy and neurodegeneration. We used the well-characterized rTg4510 mouse model of tauopathy at 5-6 months and 7-8 months of age, respectively, to study the functional impact of cortical synapse loss. The earlier age was used as a model of prodromal tauopathy, with the later age corresponding to more advanced tau pathology and presumed progression of neurodegeneration. Analysis of synaptic protein expression in the somatosensory cortex showed significant reductions in synaptic proteins and NMDA and AMPA receptor subunit expression in rTg4510 mice. Surprisingly, in vitro whole-cell patch clamp electrophysiology from putative pyramidal neurons in layer 2/3 of the somatosensory cortex suggested no functional alterations in layer 4 to layer 2/3 synaptic transmission at 5-6 months. From these same neurons, however, there were alterations in dendritic structure, with increased branching proximal to the soma in rTg4510 neurons. Therefore, in vivo whole-cell patch clamp recordings were utilized to investigate synaptic function and integration in putative pyramidal neurons in layer 2/3 of the somatosensory cortex. These recordings revealed a significant increase in the peak response to synaptically driven sensory stimulation-evoked activity and a loss of temporal fidelity of the evoked signal to the input stimulus in rTg4510 neurons. Together, these data suggest that loss of synapses, changes in receptor expression and dendritic restructuring may lead to alterations in synaptic integration at a network level. Understanding these compensatory processes could identify targets to help delay symptomatic onset of dementia.

Study on the correlation between DPP9 rs2109069 and IFNAR2 rs2236757 polymorphisms with COVID-19 mortality

Understanding the complex mechanisms of the immune system in dealing with the COVID-19 infection, which is probably related to the polymorphism in cytokine and chemokine genes, can explain the pro-inflammatory condition of patients. Therefore, in this study, the relationship between the frequency of single nucleotide polymorphisms in the two pro-inflammatory genes dipeptidylpeptidase 9 (DPP9) and interferon alpha and beta receptor subunit 2 (IFNAR2) and the severity of COVID-19 was assessed. This study involved 954 COVID-19 patients, including 528 recovered and 426 deceased patients. To investigate the polymorphisms of IFNAR2 rs2236757 and DPP9 rs2109069, we used the polymerase chain reaction with the restriction fragment length polymorphism assay. The results showed that IFNAR2 rs2236757 A allele is related to the reduced severity of the disease, whereas the incidence of DPP9 rs2109069 A allele was higher among the deceased than recovered individuals. On the other hand, in people carrying the G allele in the DPP9 gene polymorphism and the allele A in the IFNR2 gene polymorphism, the improvement of the disease was significantly higher. In conclusion, the results showed that IFNAR2 rs2236757 A allele is related to the decrease in the severity of the disease, while the frequency of DPP9 rs2109069 A allele was higher in deceased people than in recovered people. This shows the important role of genes related to inflammatory responses as well as the role of genetic variants of these genes in the severity of COVID-19.

IL-6/gp130 signaling in CD4+ T cells drives the pathogenesis of pulmonary hypertension

Pulmonary arterial hypertension (PAH) is characterized by stenosis and occlusions of small pulmonary arteries, leading to elevated pulmonary arterial pressure and right heart failure. Although accumulating evidence shows the importance of interleukin (IL)-6 in the pathogenesis of PAH, the target cells of IL-6 are poorly understood. Using mice harboring the floxed allele of gp130, a subunit of the IL-6 receptor, we found substantial Cre recombination in all hematopoietic cell lineages from the primitive hematopoietic stem cell level in SM22α-Cre mice. We also revealed that a CD4+ cell-specific gp130 deletion ameliorated the phenotype of hypoxia-induced pulmonary hypertension in mice. Disruption of IL-6 signaling via deletion of gp130 in CD4+ T cells inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) and suppressed the hypoxia-induced increase in T helper 17 cells. To further examine the role of IL-6/gp130 signaling in more severe PH models, we developed Il6 knockout (KO) rats using the CRISPR/Cas9 system and showed that IL-6 deficiency could improve the pathophysiology in hypoxia-, monocrotaline-, and Sugen5416/hypoxia (SuHx)-induced rat PH models. Phosphorylation of STAT3 in CD4+ cells was also observed around the vascular lesions in the lungs of the SuHx rat model, but not in Il6 KO rats. Blockade of IL-6 signaling had an additive effect on conventional PAH therapeutics, such as endothelin receptor antagonist (macitentan) and soluble guanylyl cyclase stimulator (BAY41-2272). These findings suggest that IL-6/gp130 signaling in CD4+ cells plays a critical role in the pathogenesis of PAH.

Lactuca sativa L. Extract Enhances Sleep Duration Through Upregulation of Adenosine A1 Receptor and GABAA Receptors Subunits in Pentobarbital-Injected Mice.

We investigated the effects of Lactuca sativa L. extracts (Lactuc) on pentobarbital-induced sleep in mice to elucidate the mechanisms underlying its impact on sleep quality. Mice were randomly assigned to five groups: control, positive control (diazepam 2 mg/kg b.w.), and three groups orally administered with Lactuc (50, 100, and 200 mg/kg b.w.). After 2 weeks of oral administration and intraperitoneal injections, the mice were killed. We found that the Lactuc-administered groups had significantly reduced sleep latency and increased sleep duration compared with the control group. Furthermore, the oral administration of Lactuc induced a significant increase in mRNA expression and protein expression of adenosine A1 receptor in the brains compared with the expressions in the control group. In addition, the Lactuc-administered groups exhibited significantly higher levels of mRNA expressions of GABAA receptors subunits α2, β2, γ1, and, γ2 in the brain tissue. Therefore, we suggest that Lactuc could be used to develop natural products that effectively improve sleep quality and duration.

Combination of bazedoxifene with chemotherapy and SMAC-mimetics for the treatment of colorectal cancer

Excessive STAT3 signalling via gp130, the shared receptor subunit for IL-6 and IL-11, contributes to disease progression and poor survival outcomes in patients with colorectal cancer. Here, we provide evidence that bazedoxifene inhibits tumour growth via direct interaction with the gp130 receptor to suppress IL-6 and IL-11-mediated STAT3 signalling. Additionally, bazedoxifene combined with chemotherapy synergistically reduced cell proliferation and induced apoptosis in patient-derived colon cancer organoids. We elucidated that the primary mechanism of anti-tumour activity conferred by bazedoxifene treatment occurs via pro-apoptotic responses in tumour cells. Co-treatment with bazedoxifene and the SMAC-mimetics, LCL161 or Birinapant, that target the IAP family of proteins, demonstrated increased apoptosis and reduced proliferation in colorectal cancer cells. Our findings provide evidence that bazedoxifene treatment could be combined with SMAC-mimetics and chemotherapy to enhance tumour cell apoptosis in colorectal cancer, where gp130 receptor signalling promotes tumour growth and progression.

Oral application of magnesium-L-threonate alleviates radicular pain by inhibiting neuro-inflammation dependent central sensitization of rats

BackgroundWe have reported neuro-inflammation is involved in radicular pain by enhancing the efficiency of pain synaptic transmission in spinal level. Recently, peers’ studies have confirmed that magnesium deficiency leads to neuro-inflammation, thus contributes to memory and emotional deficits and pain hypersensitivity in antineoplastic agents treated rats. In this study, we explore the effect of oral application of magnesium-L-threonate (L-TAMS) in radicular pain induced by lumbar disc herniation (LDH) of rats and the possible mechanisms. MethodsRat model of LDH was induced by autologous nucleus pulposus (NP) implantation. Mechanical and thermal pain thresholds were assessed by von Frey filaments and hotplate test respectively. L-TAMS was applied from drinking water at dosage of 604 mg/kg/day from 2 day before NP implantation and until the end of the experiment. Free Mg2+ content in serum and cerebrospinal fluid (CSF) was measured by calmagite chromometry. Synaptic transmission efficiency was determined by C-fiber evoked field potentials recorded by electrophysiologic recording in vivo. The activation of microglia in spinal dorsal horn was displayed by immunofluorescence staining and western blotting. The expressions of pro-inflammatory cytokines and glutamic N-methyl-D-aspartate receptor (NMDAR) subunits (NR2A, NR2B) were assessed by western blotting and enzyme-linked immunosorbent assay (ELISA) respectively. ResultsNP implantation induced mechanical allodynia and thermal hyperalgesia, accompanied by decreased Mg2+ concentration in serum and CSF which were both obscured by oral application of L-TAMS. L-TAMS inhibited spinal microglia activation and pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) expression of rats with NP. L-TAMS decreased C-fiber evoked potentials and NR2B protein level in rats with NP, which were rescued by extra intrathecal delivery of TNF-α or IL-6 or IL-1β. ConclusionsOral application of L-TAMS alleviates radicular pain by inhibiting neuro-inflammation dependent central sensitization of rats.

Limb-Clasping Response in NMDA Receptor Palmitoylation-Deficient Mice.

Proper regulation of N-methyl-D-aspartate-type glutamate receptor (NMDA receptor) expression is responsible for excitatory synaptic functions in the mammalian brain. NMDA receptor dysfunction can cause various neuropsychiatric disorders and neurodegenerative diseases. Posttranslational protein S-palmitoylation, the covalent attachment of palmitic acid to intracellular cysteine residues via thioester bonds, occurs in the carboxyl terminus of GluN2B, which is the major regulatory NMDA receptor subunit. Mutations of three palmitoylatable cysteine residues in the membrane-proximal cluster of GluN2B to non-palmitoylatable serine (3CS) lead to the dephosphorylation of GluN2B Tyr1472 in the hippocampus and cerebral cortex, inducing a reduction in the surface expression of GluN2B-containig NMDA receptors. Furthermore, adult GluN2B 3CS homozygous mice demonstrated a definite clasping response without abnormalities in the gross brain structure, other neurological reflexes, or expression levels of synaptic proteins in the cerebrum. This behavioral disorder, observed in the GluN2B 3CS knock-in mice, indicated that complex higher brain functions are coordinated through the palmitoylation-dependent regulation of NMDA receptors in excitatory synapses.

Ion transport in muscle acetylcholine receptor maintained by conserved salt bridges between the pore and lipid membrane

Pores through ion channels rapidly transport small inorganic ions along their electrochemical gradients. Here, applying single-channel electrophysiology and mutagenesis to the archetypal muscle nicotinic acetylcholine receptor (AChR) channel, we show that a conserved pore-peripheral salt bridge partners with those in the other subunits to regulate ion transport. Disrupting the salt bridges in all five receptor subunits greatly decreases the amplitude of the unitary current and increases its fluctuations. However, disrupting individual salt bridges has unequal effects that depend on the structural status of the other salt bridges. The AChR ε- and δ-subunits are structurally unique in harboring a putative palmitoylation site near each salt bridge and bordering the lipid membrane. The effects of disrupting the palmitoylation sites mirror those of disrupting the salt bridges, but the effect of disrupting either of these structures depends on the structural status of the other. Thus, rapid ion transport through the AChR channel is maintained by functionally interdependent salt bridges linking the pore to the lipid membrane.

Neurodevelopmental effects of prenatal Bisphenol A exposure on the role of microRNA regulating NMDA receptor subunits in the male rat hippocampus

Maternal bisphenol A (BPA) exposure has been reported to cause learning and memory deficits in born offspring. However, little is known that this impairment is potentially caused by epigenetic modulation on the development of NMDA receptor subunits. This study investigates the effect of prenatal BPA exposure on the hippocampal miR-19a and miR-539, which are responsible for regulating NMDA receptor subunits as well as learning and memory functions. Pregnant Sprague Dawley rats were orally administered with 5 mg/kg/day of BPA from pregnancy day 1 (PD1) until gestation day 21 (GD21), while control mothers received no BPA. The mothers were observed daily until GD21 for either a cesarean section or spontaneous delivery. The male offspring were sacrificed when reaching GD21 (fetus), postnatal days 7, 14, 21 (PND7, 14, 21) and adolescent age 35 (AD35) where their hippocampi were dissected from the brain. The expression of targeted miR-19a, miR-539, GRIN2A, and GRIN2B were determined by qRT-PCR while the level of GluN2A and GluN2B were estimated by western blot. At AD35, the rats were assessed with neurobehavioral tests to evaluate their learning and memory function. The findings showed that prenatal BPA exposure at 5 mg/kg/day significantly reduces the expression of miR-19a, miR-539, GRIN2A, and GRIN2B genes in the male rat hippocampus at all ages. The level of GluN2A and GluN2B proteins is also significantly reduced when reaching adolescent age. Consequently, the rats showed spatial and fear memory impairments when reaching AD35. In conclusion, prenatal BPA exposure disrupts the role of miR-19a and miR-539 in regulating the NMDA receptor subunit in the hippocampus which may be one of the causes of memory and learning impairment in adolescent rats.